通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体
1Centre for Medical Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences,University of Copenhagen, 2West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology,University of Ghana, 3Department of Immunology,Noguchi Memorial Institute for Medical Research, 4Centre for Medical Parasitology, Department of Infectious Diseases,Rigshospitalet
Summary
该协议的总体目标是指导如何测量自然接触恶性疟原虫感染的血清或血浆中的抗体的能力，以对抗和诱导寄生虫感染的红细胞（IEs）的噬菌体。 Plasmodium falciparum
Abstract
该协议描述了如何建立和运行一个流细胞学为基础的噬菌体噬菌体测定恶性疟原虫 – 感染红细胞 （IE） 由自然获得的IgG抗体特异性VAR2CSA。VAR2CSA是一种寄生虫抗原，它调解胎盘中可引起严重疟疾的胎盘内 IEs 的选择性固原，称为胎盘疟疾 （PM）。防止PM由VAR2CSA特异性抗体进行调解，这些抗体被认为通过抑制地盘固存和/或通过治疗吞咽病的性病来发挥作用。该检测采用体外选择的晚期同步性，以表达VAR2CSA、in vitro具有自然获得PM特异性免疫力的妇女的血浆/血清抗体，以及噬菌体细胞系THP-1。然而，该协议可以很容易地修改，以检测抗体的功能，任何寄生虫抗原存在于国际化学品表面，无论是由自然接触或接种疫苗引起的。该测定方法对疟疾抗体中量免疫的一个重要功能方面进行了简单、高通量的评价，具有良好的可重复性。因此，在评估恶性疟原虫疟疾的临床免疫力时，它很有用，恶性疟原虫是热带地区，特别是撒哈拉以南非洲地区发病率和死亡率的主要原因。
Introduction
疟疾是一种病媒传播的疾病，在感染五种不同种类的疟原虫时，由人类引起。最常见的物种是恶性疟原虫，它也负责发病率和死亡率1。疟疾临床表现从无症状或良性感染到复杂/严重疾病，后者主要发生在五岁以下儿童中。接触恶性疟原虫不会诱发无菌免疫，但生活在流行地区的个体对临床疾病的免疫力会慢慢发展。保护是年龄/暴露依赖和免疫力通常获得在生命的前5-10年2。成年妇女是一个重要的例外，因为严重的疟疾可能发生在怀孕期间，在临床介绍称为胎儿素疟疾（PM）。PM 是流产、死产、早产、低出生体重、胎儿死亡和产妇贫血的重要原因。对PM的耐药性在连续怀孕时发展 3。防止PM与获得抗体对VAR2CSA型PfEMP14，5，受感染的红细胞（IE）表面抗原，结合到软体素硫酸盐A（CSA），使国际在胎盘中固有。4,抗体调解保护执行各种功能活动（审查在,6，7），包括E的异化，以诱导吞噬。早期的体外研究表明，抗体可以通过,噬菌体8，9来限制单细胞的存在。9最近的研究表明，高浓度的噬菌体诱导抗体与更好的妊娠结果（在HIV共感染的情况下）10，11，,11表明这种效应功能在自然获得的免疫反应的相关性。
在这里，我们提出了一个协议，测量存在于人类血浆/血清中的抗体的这一功能in vitro，使用体外培养的IES表达VAR2CSA与单细胞线THP-1。与早期的显微镜方案8相比,,，该测定方法以前曾使用过,,,11、12、13、14、15、16、17、18，被认为是一种改进12和更容易的方法，因为它允许使用较小的抗体体积在一次运行中测试更多的抗体样本，并避免繁琐和偏颇的18显微镜计数。17,1113141516尽管该测定方法已被多个实验室使用，而且执行过程非常简单，但它需要仔细规划和准备，因此，详细的协议将允许缺乏经验的实验室和研究人员应用该测定。例如，我们使用表示 VAR2CSA 的后期同步 IE 与从自然获得 PM 特异性免疫力的妇女收集的血清中存在的抗体进行结果化。然而，该协议可以很容易地修改，以检测抗体的功能，任何寄生虫抗原存在于国际化学品表面，无论是由自然接触或接种疫苗引起的。
Protocol
用于此处结果的人体血清样本在另外一项研究19中收集。馆藏由加纳大学野口医学研究所机构审查委员会（研究报告038/10-11）和丹麦首都大区区域研究伦理委员会（协议H-4-2013-083）批准。 1. 寄生虫文化 注：遵守当地有关人类病原体处理的规定。 根据寄生虫培养基20前所述的标准协议维持恶性疟原虫（参见20</su…
Representative Results
在这里，我们详细介绍了一个协议，以前描述31，并使用11，12，13，14，15，16，17，18,14,15,16,17,1811,12,测量抗体的能力，目标为P.恶性疟原虫的表，诱导蛋白石化和吞噬的THP…
Discussion
这里提出的协议先前已经描述，并使用,12，15，17，31,31测量抗体的能力，针对P.恶性疟原虫的表层，以诱导蛋白位和吞噬的THP-1细胞。15,此处提供的结果侧重于生活在恶性疟原虫流行地区的妇女的血浆/血清中自然获得的VAR2CSA特异性抗体。VAR2CSA 是一种参与 IEs 的塞盘固存的 PfEMP1 类型，是 …
Divulgations
The authors have nothing to disclose.
Acknowledgements
迈肯·维斯蒂感谢出色的技术援助。这项工作部分由丹麦外交部的赠款（MAVARECA II;17-02-KU）供资，由丹妮达研究金中心管理。资助者在研究设计、数据收集和分析、决定出版或准备手稿方面没有作用。
Materials
| 96 well cell culture plates, round bottom with lid | Corning | 3799 | Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use |
| AlbuMAX-II | Gibco | 11021-037 | |
| AlbuMAX-II (5%) | – | – | 5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886) |
| Anti-Red Blood Cells antibody | Abcam | ab34858 | Prepare 2��l aliquots and freeze a -20°C. Use one aliquot per experiment. |
| DPBS | Sigma | P8622 | |
| Dynabeads Protein G | Invitrogen | 10003D | |
| Ethidium bromide solution | Sigma | E1510 | Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light |
| FC500 flow cytometer | Beckman Coulter | Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used. | |
| Fetal Bovine Serum (FBS) | Gibco | 10099-141 | Heat inactivate before use. |
| FITC mouse anti-human CD16 | BD Biosciences | 555406 or 556618 | |
| FITC mouse anti-human CD32 | BD Biosciences | 552883 | |
| FITC mouse anti-human CD64 | BD Biosciences | 555527 | |
| FlowLogic software | Inivai technologies | Any flow cytometry analysis can be used, for example FlowJo or Winlist | |
| Gentamicin (10mg/mL) | Sigma | G1272 | |
| Hypoxanthine | Sigma | H9377 | |
| L-glutamine (200mM) | Sigma | G7513 | |
| Lysing solution | – | – | 15mM NH4Cl, 10mM NaHCO3, 1mM EDTA |
| MACS CS-column and accesories | Miltenyi Biotec | 130-041-305 | |
| Parasite culture medium | – | – | 2mM L-glutamine (Sigma, G7513), 50µg/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886) |
| Penicillin/Streptomycin (10000U and 10mg/mL) | Sigma | P0781 | |
| RPMI-1640 medium | Sigma | R5886 | |
| THP-1 culture medium | – | – | 10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886) |
| Vario MACS magnet | Miltenyi Biotec |
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Citer Cet Article
Quintana, M. d. P., Anabire, N. G., Hviid, L. Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay. J. Vis. Exp. (162), e61538, doi:10.3791/61538 (2020).